Process for producing 16alpha-hydroxy-steroids using nocardia it alica



United States Patent 0 3,285,829 PROCESS FOR PRODUCING ISa-HYDROXY-STEROIDS USING NOCARDIA I TALICA Alba Maria Amici, Maria Luisa Bianchi,Renato Modelll, Celestino Spalla, Aurelio Di Marco, and MarcelloGaetani, all of Milan, Italy, assignors to Societa Farmaceutici Italia,Milan, Italy, a corporation of Italy No Drawing. Original applicationJuly 13, 1962, Ser. No. 209,784, now Patent No. 3,188,325, dated June 8,1965. Divided and this application Aug. 26, 1964, Ser. No. 398,140

Claims priority, application Italy, July 17, 1961, 13,227/ 61; Mar. 14,1962, 5,088/ 62 13 Claims. (Cl. 19551) This application is a division ofapplication Serial No. 209,784, filed July 13, 1962, now Patent No.3,188,325.

Our invention relates to a new method for the preparation of 1fiahydroxy-steroids. Our invention has as an object a microbiologicalprocess to introduce a hydroxy 3,285,829 Patented Nov. 15, 1966 80%) andhigh steroid concentration (0.1%) in the culture broth.

Description and identification of the strain.-The new microorganismNocardia italica isolated from a soil sample has the followingmorphologic, cultural and biochemical characteristics.

On the usual culture media, the microorganism forms at first aspread-out mycelium with very branched, nonseptate hyphae. After 3-4days the formation of transversal septa is observed and afterwards themyceliu-m fragments into portions having various shapes and lengths(4-8 1) The hyphae thickness ranges from 0.5 to 0.7,u. On liquid media,the fragmentation occurs within 2 or 3 days; V and Y forms are frequent,while coccoid forms are absent. No aerial mycelium is observed in anymedium. The mycelium is gram-positive and partially acid fast.

In the following table the cultural characteristics are reported; theobservations have been carried out after 10-1520-25 days of incubationat 28 C. The cultures group into the l6a-p0sition of a steroid molecule,using 20 have been repeated six times for each type of medium.

' TABLE I Vegetative Mycelium Culture medium Soluble Pigments RemarksGrowth Color Czapek Agar Slight Colorless Yeast Agar. Abundant wrinkledgrowth... Yellowish Malt Agar" do "do Potato Agar Egg-yellowish Abundantgrowth with relieved folds Scattered small colonies Abundant, wrinkled,pasty consistence.

Jensen Agar Nutritive Agar with glycerol.

Bonnet Agar Abundant wrinkled growth, Orange-yellowish.

pasty consistence. Glucose asparagine Agar Good, uniform growth, pastyEgg-yellowish is consistence. Sabouraud Agar. Abundant, wrinkled growth,Brown-yellowish pasty consist. Starch Agar Good, uniform Orange it Ithydrolyzes abundantly. Yeast broth Flaky colonies at the bottomYellowish Peptone broth with potassium Flaky colonies at the bottomNitrates not reduced.

nitrate. end. Potato plugs Abundant with large folds Egg-yellowish MilkSlight ring-pellicle, Ycllowish Peptonlzatlon and coagulation in 15days. Slight fluidifi-cation.

the new microorganism Nocardz'a italica n. sp. A further object is theproviding of some new 16a-hydroxy-17amethyl-androstenols of the normaland 19-nor-series, obtained by the process of the invention.

16oc-hydroxy-steroids, which are well known in the literature both asintermediates for preparing therapeutically useful substances and asproducts having a high antiphlogistic activity, are prepared byemploying some microorganisms. The most important of thesemicroorganisms are: Streptomyces roseocromogenus (South African patentapplication No. 3,300/ 58), Streptomyces ATCC 13278 and StreptomycesATCC 13279 (Indian Patents No. 67,019 and No. 69,056), Streptomyccsviridis, Streptomyces olivacetrs, Streptomyces argenteo-lus (US. Patents No. 2,709,705 and No. 2,855,343). The literature reports otherstrains able to hydroxylate the 16a-position, namely Actinomyceslavendulae, Pestallotia fwnerea, Didimclla vodaldi, and Strepmmycesmediociaious, Streptomyces halstedii.

The new microorganism employed in the process of the present invention,named Nocardia italica n. sp., which has been deposited with theNational Collection of Industrial Bacteria receiving the index numberN.C.I.B. 9386 and at the Institute of Microbiology of Rutgers Universityreceiving the number 3856, belongs to the genus Nocmrdia trevimn whosecapacity of hydroxylating a steroid in the 16a-position has beenhitherto unknown. The use of Nocardia italica gives high transformationyields (70- The biochemical characteristics of the microorganismNocardia italica are the following:

Gelatine: slow and slight fiuidification;

Nitrates: no reduction to nitrites;

Milk: coagulation and peptonization;

Starch: abundant hydrolysis; and

Acids production: positive from maltose, d-xylose, d-mannose, mannitol,glycerol, glucose, levulose; negative from lactose, adonitol,d-sorbitol, -arabinose, saccharose, trehalose, rafiinose, esculine.

The description or the tested microorganism corresponds to that of thegenus Nocardia trevisan, which has been referred to in Bergeys Manual ofdeterminative Bacteriology (seventh edition 1957, pages 713-715) so thatwe may conclude that the microorganism of the present invention belongsto the genus Nocardia trevisan.

The analytic key of the species of genus Nocardia Waksman and Henrici(Waksman and Lechevalier: Guide to liquefies gelatine, does not showpink or red colored mycelium in any tested media and does not formaerial mycelium on potato plugs. Therefore, we conclude that the testedmicroorganism has neither been isolated nor described before.

The stock cultures of N. italica may be stored by lyophilization, milkor milk serum being the suspending medium. It may also be kept bysuccessive transfers on glu- 'cose potato agar.

Our invention provides a microbiological process of preparinglfia-hydroxy-steroids which comprises treating a steroid compound havinga methylenic group in the 16- position with a culture produced byfermenting the new microogranism Nocardia italica n. sp. or a mutantthereof in a sterilized liquid nutritional medium containing assimilablesources of carbon and nitrogen at from 24 to 30 C. at a pH of from 6 to7.5, and extracting the 16mhydroxy-steroid produced.

The-process of our invention may be applied to a great number ofsteroid, compounds, more generally to all steroid compounds having from19 to 21 carbon atoms which may contain in their molecules, for example,one or more double bonds in the 1-, 4-, 6- or 9 11)-positions, halogenatoms in the 6- or 9-position, one or more hydroxy or alkoxy groups inthe 9-, 11-, 17- or 21-positions, one or more keto groups in the 3-,11-, 17-, or 20-positions, one or more alkyl groups in the 6- or17-positions.

The new compounds of the invention are:

16ot-hydroxy-steroid having anticancer activity of the general formula:

om p R Typical examples of steroid compounds which can be subjected tothe fermentative process by using N. italz'ca are: progesterone,cortisone, hydrocortisone, testosterone,

Reichsteins Compound S, desoxycorticosterone, 9zx-flLlOIO-hydrocortisone, 9a-fluoro-prednisolone, 4-androstene-3,17- dione,9a-chloro-hydrocortisone, 9a-bromo-hydrocortisone,ll-epi-hydrocortisone, Qa-HUOIO-COIlLiSOIlC, 9ot-methoxy-cortisone,6u-fluoro-hydrocortisone, 6a-methyl-hydrocortisone, 4,6pregnadiene-l1p,17a,21-triol-3,20-dione, 17a methyl-19-nor-testosterone,4-hydroxy-17a-methyltestosterone,4-hydroxy-17a-methyl-19-nor-testosterone.

The process of our invention introduces a hydroxy group into thelfioc-position of steroids and consists in fermenting N. italica insuitable media and conditions, and submitting the starting16-desoxy-steroid to the action of the microorganism or to the enzymesthereof. Said steroid may be added to the cultures either at thebeginning, or during or at the end of growth, either in crystalline formor dissolved in suitable solvents such as methyl alcohol, ethyl alcohol,acetone or dimethylformamide for example.

The culture media consist of a source of carbon, of nitrogen and ofinorganic salts. The carbon source may be constituted by starch,dextrin, saccharose, glucose, maltose, glycerin, vegetable oils, cerealor legume flour, corn steep liquor and/or other substances usuallyemployed. The nitrogen source, besides the above-cited complexsubstances containing nitrogen, may be constituted by casein, ammoniasalts, peptones, meatand fish-flour or other substances usuallyemployed. The inorganic salts may be sodium, potassium, magnesium,sulfates, phosto add surfactants, such as available under the trademarksTween 80 and Span.

The microorganism grows in submerged and aerated conditions, in shakeflasks or in fermenters at a temperature ranging from 24 to 30",preferably at 28 C. The pH of the medium should range between 6 and 7.5,preferably at 6.8. During fermentation the pH is quite constant.

The transformation of the starting 16-desoxy-steroid into thecorresponding lfiwhydroxy-derivative during fermentation is checked bychromatography on thin layers. The chromatography on thin layers isdescribed in the literature by EgonStahl (Pharmac. Weekbl-ad 92 1957,page 829; Chemiker Ztg. 82 1958, page 323) and by N. J. D. Van Dam etal, (I. Chromatography 4 1960, page 26). Chromatography on thin layersis also very suitable for a quantitative analysis by comparing the spotintensity with that of standard steroid solution of knownconcentrations. The steroid substance contained in the culture broth(both the reacted products and the unreacted starting material) isrecovered by known extraction processes. Preferably the extraction iscarried out in the following manner.

The fermentation broth is mixed with a siliceous earth material, such asSupercel or Celite (registered trademarks) and the resulting mixture iswarmed up to 50- 60 C. for a few minutes, whereupon the whole isfiltered, and the filtration cake is washed with water at 50 C. anddiscarded. The filtrate is extracted with an organic solvent, such asethyl acetate, chloroform or methylene dichloride, the extracts arecombined and washed with a saturated solution of sodium bicarbonate,then with water to neutrality. The solvent is evaporated and thetransformation product is recovered in the usual manner. Thetransformation product often crystallizes during concentration. It maybe convenient to reorystallize the residue from acetone-petroleum etheror from other organic solvents. In other cases, column chromatographyover silica gel may be used to obtain the compound in pure form.

Instead of submitting the crude products of the present invention to theabove-cited purifying process, they may be acylated in 4,16-position orin the 16-position with the anhydride or the chloride of an aliphatic-,cycloaliphatic-, aromatic-acid having from 1 to 9 carbon atoms in theoptional presence of tertiary amines such as pyridine or the analoguesthereof, dimethylaniline, trim'ethylamine, and then they are isolatedand purified. Typical examples of acyl derivatives, prepared accordingto the present invention, are the derivatives of the following acids:acetic, propionic, cyclopentylpropionic, benzoic, phenylpropionic.

We have also found, and this is still a further object of our invention,that submitting some 17u-methy1-androstenols of the normaland19-nor-series to the above-menphates, chlorides and calciumcarbonate. It is also useful tioned fermenting process with Nocardiaitalzczz n. sp., the androgenic properties of the starting16-desoxy-steroid are so remarkably reduced as to make them negligible,while the 16a-hydroxysteroids obtained show a remarkable activity ininhibiting the experimental tumors.

7 The products of the invention which have proved useful in the therapyof tumors are: 4,16u-dihydroxy-17amethyl-testosterone and its4,16-diacyl-derivatives, 4,16- dihydroxy-17ot-methyl-l9-nor-testosteroneand its 4,16- diacyl-derivatives,16a-hydroxy-l7u-methyl-19-nor-testosterone'and its l6-acyl-derivatives.The starting 16-desoxysteroid for the preparation of4,l6u-dihydroxy-l7a-methyl-testosterone is4-hydroxy-17a-methyl-testosterone (described in the British Patent No.848,288), while the starting 16-desoxy-steroid for the preparation of4,160- dihydroxy-l7a-methyl-19-nor-testosterone is 4-hydroxy-17a-methyl-19-nor-testosterone (described in the British Patent No.888,665) and the 16-desoxy-steroid for the preparation of16a-hydroxy-17a-methyl-19-nor-testosterone is17a-methyl-19-nor-testosterone. Among the abovementioned compounds theonly product known in the literature is 16ahydroxy-17a-rnethyl-19-nor-testosterone,

described by Seymour and E. W. Cantrall (J. Org. Chem.

26, 1961, page 3560), who report only a very Weak androgenic propertybut without any mention of an anticancer power.

These products have an anticancer activity and have shown especiallyvaluable in inhibiting E-hrlichs adenocarcinoma and Ehrlichs ascitistumor in mice. As will be further demonstrated, the products of thepresent invention, administered to mice, inoculated with Ehrlichsadenocarcinoma or Ehrlichs ascitis tumor cause the regression of tumorwithout any deleterious effect. They are administered preferably by asubcutaneous route as a suspension or solution in a suitable organicdiluent such as polyethylene glycols, mineral oils, complex esters andtensioactive.

The following examples serve to illustrate, but are not intended tolimit, the present invention.

EXAMPLE, 1

A culture of N. italica aged 5 days on glucose-potatoagar was employedto inoculate two 300 cc. Erlenmeyer flasks each containing 60 cc. of thefollowing medium:

Casein 2 Dextrin 20 Corn steep 3 Calcium carbonate, CaCO 4 Ammoniumsulfate, (NH SO 1 Potassium hydrogen phosphate, K HPO 0.1 Glucose 1O Tapwater 1000 pH 6.6

Sterilization: 120 C. for 20' minutes.

The flasks were incubated at 28 C. for 40 hours on a rotary shaker witha range of 6 cm. at 220 r.p.m. 6 cc. of the culture thus obtained wereused to inoculate ten 300 cc. flasks, each containing 60 cc. of thefollowing medium:

Casein g 2 Dextrin g 20 Corn steep g 15 Calcium carbonate, OaCO 4Ammonium sulfate, (NH SO g 1 Potassium hydrogen phosphate, K HPO g 0.1Glucose g Tween 80 cc 0.5 Soya oil cc 20 Tap Water cc 1000 pH 6.6

Sterilization: 120 C. for 30 minutes.

60 mg. of 9u-fluoro-hydrocortisone, dissolved in 0.5-

cc. of dimethylformamide were then added to the flasks. The flasks wereincubated at 28 C. on a rotary shaker similar to that described for thepreparation of the vegetative culture.

A chromatographic test, carried out after 3 days of incubation, showedthe disappearance of the starting product and the formation of a producthaving an Rf equal to that of 16a-hydroxy-9a-fluoro-hydrocortisone. Thecontents of the flasks were then combined, and 100 g. of Celite Wereadded thereto and the resulting mass was warmed up to 60 C. withstirring for 10 minutes. The mycelium cake was then filtered and washedwith 100 cc. of water at 60 C. The filtrate was extracted 3 times withethyl acetate. The ethyl acetate extracts were combined, washed oncewith a saturated solution of sodium bicarbonate, then twice withdistilled Water. The organic extract was evaporated in vacuo to reducethe volume to about 150 cc. of residual solution, which was transferredinto a smaller flask and evaporated to a volume of 30 cc.; at this stagean abundant crystallization took place. The flask was kept in arefrigerator for 20 hours, then the product was filtered and washed withcold ethyl acetate. 0.460

6 g. of 16a-hydroxy-9a-fluoro-hydrocortisone were obtained, melting at240-245 C.; [a] =|1O0 (c.='1 in meth anol).

Upon recrystallization from methanol ethyl acetate, 0.400 g. of16a=hydroxy-9a-fluoro-hydrocortisone were obtained, melting at 248250C.; [a] =-l-l05 (c.=l in methanol). Yields EXAMPLE 2 The fermentationwas carried out as in Example 1 while using a medium of the followingcomposition:

Distillers solubles g 10 Meat extract g r 5 Sodium chloride, NaCl g 2.5Glucose g 15 Emulsifier, Tween 80 cc 0.5 Tap water cc 1000 pH 6.7

Sterilization: C. for 20 minutes.

A chromatographic test showed the disappearance of the starting productafter 4 days of incubation. A prodnot with an R equal to that ofl6oc-hYdIOXY-9a-flll0l'0- hydrocortisone was obtained. Extraction withethyl acetate and subsequent concentration until crystallization beginsgive pure 16a-hydroxy-9a-fluoro-hydrocortisone in a yield of 75%.

EXAMPLE 3 3 liters of the vegetative medium as in Example 1 weresterilized at 120 C. for 60 minutes in a 5 l. laboratory fermenter. Theywere then inoculated with 30 cc. of culture broth in a flask asdescribed in Example 1 and thereafter incubated for 24 hours at 28 C.with stirring with a four-paddle propeller at a rate of 400 r.p.m. andat an aerating rate of 3 liters per minute.

5.4 liters of the fermentation medium as described in Example 1 aresterilized in a 10 l. laboratory fermenter and inoculated with 600 cc.of vegetative medium. 6 g. of 9a-fluoro-hydrocortisone dissolved in 35cc. of dirnethylformamide were added under sterile conditions to themedium and incubated at 28 C. with stirring with a four-paddle propellerat a rate of 450 r.p.m. and under an aerating rate of 5 liters perminute.

A chromatographic test showed the disappearance of the starting compoundafter 65 hours of fermentation. Extraction with ethyl acetate andsubsequent concentration gave 16or-hydroxy-9a-fluoro-hydrocortisone in ayield of 80%.

EXAMPLE 4 The fermentation was carried out as in Example 1 using9a-fluoro-prednisolone as the starting material. As soon as thechromatographic test showed that 9u-fluoroprednisolone was completelytransformed, the usual extraction was performed, whereby16a-hydroxy-9a-fluoroprednisolone melting at 248-250 C. was obtained. Onrecrystallization from ethyl acetate-methanol the pure product meltingat 260262 C. was obtained,

[ ]D =+68 (c.=1 in methanol) EXAMPLE 5 The fermentation was carried outas in Example 1 by employing 60 mg. of hydrocortisone as the startingsteroid, 16m-hydroxy-hydrocortisone melting at 225-230 C. (crystallizedfrom ethyl acetate) was obtained;

[a] =+1120 (c.==l in methanol On recrystallization from ethyl acetatel6a-hydroxyhydrocortisone melting at 232-235 C. was obtained.

EXAMPLE 6 The fermentation was carried out as in Example 1 usingprogesterone (60 mg) as the starting steroid. On recrystallization fromethyl acetate 16u-hydroxy-progesterone melting at 212214 C. wasobtained,

EXAMPLE 8 The fermentation was carried out as in Example 1 usingReichsteins Compound S as the starting material. Upon extraction withethyl acetate, evaporation to dryness and crystallization fromacetone-ether 4-pregen- 16oz,l7rx,2l-t1i0l-3,20-di0n8 was obtained;melting point 235-240 C.; [a] =+93 (0.:1 in methanol).

EXAMPLE 9 The fermentation was carried out as in Example 1, using 60 mg.of desoxycorticosterone as the starting material. Upon extraction withethyl acetate and concentration to a small volume,16ot-hydroxy-desoxycorticosterone separated; melting point 180190 C.

On crystallization from dichloroethane pure16mhydroxy-desoxycorticosterone melting at 202204 C. was obtained; [a]+l12 (0.: in methanol).

EXAMPLE 10 The fermentation was carried out as in Example 1 using 60 mg.of desoxycorticosterone acetate as the starting material. Uponextraction with ethyl acetate and crystallization from dichloroethane,16a-hydroxy-des0xycorticosterone, melting at 202-204 C., was obtained.

In this case, Nocardia italica, besides introducing a hydroxy group intothe 16a-position, also saponified the 21-acetate-group.

EXAMPLE 11 The preparation was carried out in the same way as 8 EXAMPLE13 4,16a-dihydr0xy-1 7ot-methy l-testosterone The preparation wascarried out in the same way as in Example 1, from4-hydroxy-17otmethyl-testosterone. 4,16a-dihydroxy-17a methyltestosterone was obtained, melting at 226228 C.; [a] :+36.2 (c.=1 indiox- .ane);

infir 2 7 tta. 393

By reacting said compound. with an acylating agent in known manner thecorresponding 4,16-diacyl-derivatives were obtained. In this manner4,16-diacetate, 4,16- dipropionate, 4,16-dibenzoate, and other4,16-diacylderivatives were obtained.

EXAMPLE 14 4,16wdihydr0xy-1 7ot-methyl-1 9-inor-testotrterone Thepreparation was carried out in the same way as in Example 1 from4-hydroxy-171x-methyl-19-nor-testosterone.4,16a-dihydroxy-17a-methyl-19-nor testosterone was obtained, whichcrystallized with difiiculty. By acetylating with acetic anhydride inpyridine, in known manner, the corresponding 4,16-diacetate wasobtained, melting at 156-158" C.; [a] 1 c.=1 in dioxane);

max.

at 246 m Eff EXAMPLE 15 Pharmacology In the following table theandrogenic and anticancer activities of4,16ot-dihydroxy-17ot-rnethyl-19-nor-testosterone, or4,16ot-dihydroxy-17a-methyl-testosterone and of1ot-hydroxy-17u-methyl-19-nor-testosterone are reported in comparisonwith the starting 16-desoxy-steroids. Their androgenic activity wasdetermined according to the method of Hershberger et al. (Pros. Soc.Exp. Biol, and Med. 83 1953, page 175) and the anticancer activity wastested on Ehrlichs adenocarcinoma. The values of the androgenic activityare referred to testosterone propionate, conventionally taken as equalto 100, while the values of the anticancer activity are listed aspercentage of inhibiting the increase of Ehrlichs adenocarcinomaexperimentally induced in mice.

The products were administered subcutaneously to mice as describedabove. The table shows that 160:- hydroXy-steroids, preparedv accordingto the present invention, present a negligible androgenic activity andan anticancer activity remarkably higher than that of the starting16-desoxy-steroids.

TABLE II Steroids Activity Androgenic Anticancer4-hydroxy-17a-mctl1y1-19mor-testosterone4,ltia-dihydroxy-17a-methy1-19-nor-testosterone4-hydroxy-17a-methyltestosterone4,16a-dihydroxy-17w1nethy1-testosterone. 17a-metllyl-19-n0r-testosterone16a-hydroxy-17a-methyl-l9-nor-testosterone 25 45.8%300 mg., kg./day 10days.

39.2%225 mg./kg./day 6 days. 37% 135 mg./kg./day 7 days. 19.4%300mg./kg./dayX9 days. 20%143 mg./kg./day 3 days. 38.6%275 mg./kg./day l0days. 36.8%92.5 mg./kg./day l0 days.

Very sligl1t- 22.5 Very Slight. 17.4 Very slight in Example 11,differing only in the extraction technique. More exactly, the extractionof the fermentation broth was carried out with methyl-isobutyl-ketonewithout previously removing the mycelium, then by chromatographing thedry extract over Florisil, an activated magnesium silicate:16a-hydroxy-17a-methyl-19-nor-testosterone was eluted with ethyl-ethercontaining 10% of acetone. The 16a-acetyl-derivative, obtained in knownmanner with acetic anhydride in pyridine, melts at 177-180 C; [w] 2 ::17(c.=1 in dioxane);

Ami? 240 mp; Eff 473 and a pH between 6 and 7.5.

3. A process for producing a 16ot-hydroxy-steroid which comprisestreating a 16-desoxy-steroid selected from the pregnane and androstaneseries With the microoragnism Nocardia italica n. sp. under aerobic andsubmerged conditions at a temperature of about 28 C. and a pH of about6.8.

4. A process for producing 9a-fiuoro-16a-hydroxyhydrocortisone whichcomprises treating 9a-fluoro-hydrocortisone with the microorganismNocardia italica n. sp. under aerobic and submerged conditions.

5. A process for producing 16oa-hydroxy-progesterone which comprisestreating progesterone with the microorganism Nocardia italica n. sp.under aerobic and submerged conditions.

6. A process for producing 16a-hydroxy-hydroc0rtisome which comprisestreating hydrocortisone with the microorganism Nocardia italica n. sp.under aerobic and submerged conditions.

7. A process for producing 16a-hydroxy-testosterone which comprisestreating testosterone With the microorganism Nocardia italica n. sp.under aerobic and submerged conditions.

8. A process for producing 4-pre|gnene-16a,17a,21- trio1-3,20-dionewhich comprises treating Reichsteins Compound S with the microorganismNocardia italica n. sp. under aerobic and submerged conditions.

9. A process for producing l6a-hydroxy-desoxy-corticosterone whichcomprises treating desoxy-corticosteroneQl-acetatre with themicroorganism Nocarclia italica n. sp. under arerobic and submergedconditions.

10. A process -for producing t-flllO1'O-160t-hYd1'OXyprednisolone whichcomprises treating 9ot-fluoro-prednisolone With the microorganismNocatrdia italica n. sp. under arerobic and submerged conditions.

11. A process for producing 4,16oc-dihY-dIOXY-17otmethyl-testosteronewhich comprises treating 4-hydroxy- Not-methyl testosterone with themicroorganism Nocardia italica n. sp. under aerobic and sub-mergedconditions.

12. A process for producing4,16u-dihydroxy-17amethyl-19-nor-testosterone which comprises treating4- hydoxy-17a-methy1-19-nor-testosterone with the microorganism Nocardiaitalica n. sp. under aerobic and submerged conditions.

13. A process for producing 16tx-hydroxy-17o-methy1- 19-nor-testosteronewhich comprises treating the 17amethyl-l9-nor-testosterone with themicroo ganism N0- cardia italica n. sp. under aerobic and submergedconditions.

References Cited by the Examiner UNITED STATES PATENTS 2,855,343 10/1958Fried et a1 51.013 3,047,569 7/1962 Holmlund et a1. 195-51 A. LOUISMONACELL, Primary Examiner.

D. M. STEPHENS, Assistant Examiner.

1. A PROCESS FOR PRODUCING A 16A-HYDROXY-STEROID WHICH COMPRISESTREATING A 16-DESOXY-STEROID SELECTED FROM THE PREGNANE AND ANDROSTANESERIES WITH THE MICROORGANISM NOCARDIA ITALICA N. SP. UNDER AEROBIC ANDSUBMERGED CONDITIONS.